Biology subjects

Surtees, J. A.

Publications and source records attributed to Surtees, J. A..

5 recordsLinked to original sources

bioRxiv2024

Msh2-Msh3 DNA-binding is not sufficient to promote trinucleotide repeat expansions in Saccharomyces cerevisiae

Mismatch repair (MMR) is a highly conserved DNA repair pathway that recognizes mispairs that occur spontaneously during DNA replication and coordinates their repair. In Saccharomyces cerevisiae, Msh2-Msh3 and Msh2-Msh6 initiate MMR by recognizing and binding insertion deletion loops (in/dels) up to [~] 17 nucleotides (nt.) and base-base mispairs, respectively; the two complexes have overlapping specificity for small (1-2 nt.) in/dels. The DNA-binding specificity for the two complexes resides in their respective mispair binding domains (MBDs) and have distinct DNA-binding modes. Msh2-Msh3 also plays a role in promoting CAG/CTG trinucleotide repeat (TNR) expansions, which underlie many neurodegenerative diseases such as Huntingtons Disease and Myotonic Dystrophy Type 1. Models for Msh2-Msh3s role in promoting TNR tracts expansion have invoked its specific DNA-binding activity and predict that the TNR structure alters its DNA binding and downstream activities to block repair. Using a chimeric Msh complex that replaces the MBD of Msh6 with the Msh3 MBD, we demonstrate that Msh2-Msh3 DNA-binding activity is not sufficient to promote TNR expansions. We propose a model for Msh2-Msh3-mediated TNR expansions that requires a fully functional Msh2-Msh3 including DNA binding, coordinated ATP binding and hydrolysis activities and interactions with Mlh complexes that are analogous to those required for MMR. Article SummaryThe mismatch repair (MMR) protein complex Msh2-Msh3 promotes trinucleotide repeat (TNR) expansions that can lead to neurodegenerative diseases, while the Msh2-Msh6 complex does not. We tested the hypothesis that Msh2-Msh3s specific DNA binding activity is sufficient to promote TNR expansions, using a chimeric MSH complex in vivo and in vitro. We found that the Msh2-Msh3-like DNA-binding was not sufficient to promote TNR expansions. Our findings indicate that Msh2-Msh3 plays an active, pathogenic role in promoting TNR expansions beyond simply binding to TNR structures.

genetics↗

bioRxiv2022

Msh2-Msh3 interferes with DNA metabolism in vivo

Mismatch repair (MMR) is a highly conserved DNA repair pathway that safeguards the genome from errors in DNA replication. In Saccharomyces cerevisiae, two MutS homolog (Msh) complexes, Msh2-Msh3 or Msh2-Msh6, initiate MMR. Msh2-Msh3, the focus of this study, recognizes and directs repair of insertion/deletion loops (IDLs) up to ~17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR), and the DNA damage response. Msh2-Msh3 also promotes genome instability through trinucleotide repeat (TNR) expansions. This non-canonical activity is likely an unfortunate consequence of Msh2-Msh3s intrinsic ability to bind a wide range of DNA structures, including those formed with single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5 ssDNA flap structures interfered with the in vitro binding and cleavage activities of the flap endonuclease Rad27 (Fen1 in mammals), which promotes 5 ssDNA flap processing during Okazaki fragment maturation (OFM) and long-patch base excision repair (LP-BER). Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and LP-BER in vivo, consistent with the hypothesis that protein abundance and Msh3 ATPase activities are key drivers of Msh2-Msh3-mediated genomic instability.

genetics↗

bioRxiv2022

Challenges in Improving Genomic Literacy: Results from National and Regional Surveys of Genomic Knowledge, Attitudes, Concerns, and Behaviors

PurposeInformation about genomics is increasingly available to mainstream society, with more and more emphasis on using genomic information to make health care decisions. To determine how prepared people are to use this knowledge to make critical health-related decisions, we assessed the publics level of genomic literacy and whether this knowledge affects their engagement in behaviors related to genomics, such as getting genetic testing. MethodsA survey assessing perceived and actual knowledge, attitudes, concerns, sources of information, and behaviors related to genomics was administered to national and regional samples of participants. A hierarchical linear regression tested whether knowledge and attitudes predicted engagement in behaviors related to genomics. ResultsParticipants had good basic knowledge of genetics, though they were less familiar with the term "the human genome." They also displayed positive attitudes towards genomic research, despite expressing many concerns. Both greater knowledge and more positive attitudes significantly and independently predicted greater engagement in genetic testing and other related proactive health behaviors. ConclusionKnowledge and concerns about genomics impact the publics ability and willingness to obtain genetic testing and engage in other proactive health behaviors. The publics genomic literacy could be enhanced by integrating their knowledge (e.g of DNA) with broader concepts (e.g. the human genome and genomics) and how they relate to health. Future research is needed on interventions that do this, to improve the publics genomic literacy through relationships that build trust

scientific communication and education↗

bioRxiv2021

Complex Mutation Profiles in Mismatch Repair and Ribonucleotide Reductase Mutations Reveal Novel Repair Substrate Specificity of MutS Homolog (MSH) Complexes

Determining mutation signatures is standard for understanding the etiology of human tumors and informing cancer treatment. Multiple determinants of DNA replication fidelity prevent mutagenesis that leads to carcinogenesis, including the regulation of free deoxyribonucleoside triphosphate (dNTP) pools by ribonucleotide reductase (RNR) and repair of replication errors by the mismatch repair (MMR) system. We identified genetic interactions between rnr1 alleles that elevate dNTP levels and MMR. We then utilized a targeted deep-sequencing approach to determine mutational signatures associated with MMR pathway defects. By combining rnr1 and msh mutations to increase dNTP levels and alter the mutational load, we uncovered previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct repair of G/C single base deletions in GC runs, while Msh2-Msh6 specifically directs repair of substitutions at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds. Finally, we observed that the mutation profiles in double mutants were not necessarily an additive relationship of mutation profiles in single mutants. Our results have implications for interpreting mutation signatures from human tumors, particularly when MMR is defective.

genetics↗

bioRxiv2020

Targeted Next Generation Sequencing in Saccharomyces cerevisiae Reveals Complex Mutation Profiles in the Presence of Mismatch Repair and Ribonucleotide Reductase Mutations that Compromise the Fidelity of DNA Replication

Distinct mutation signatures arise from environmental exposures and/or from defects in metabolic pathways that promote genome stability. The presence of a particular mutation signature in a cell or a tumor can therefore predict the underlying mechanism of mutagenesis, which, in practice, may be clinically important. These insults to the genome often alter dNTP pools, which itself impacts replication fidelity. Therefore, the impact of altered dNTP pools should be considered when making mechanistic predictions based on mutation signatures. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to define information-rich mutational profiles associated with distinct rnr1 backgrounds that alter replication fidelity by elevating dNTP levels.. The mutation spectra of rnr1Y285F and rnr1Y285A alleles were characterized previously; our analysis was consistent with this prior work but the sequencing depth achieved in our study allowed a significantly more robust and nuanced computational analysis of the variants observed, generating profiles that integrated information about mutation spectra, position effects, and sequence context. This approach revealed novel, genotype-specific mutation profiles in the presence of even modest changes in dNTP pools. Furthermore, we identified broader sequence contexts and specific nucleotide motifs that influenced variant profiles in different rnr1 backgrounds, which allowed us to make specific mechanistic predictions about the impact of altered dNTP pools on replication fidelity.

genetics↗